Soil fungus Xylaria sp. Y01-based cytochalasin and application thereof

Abstract

The invention provides cytochalasin based on soil fungi Xylaria sp. Y01 and application of the cytochalasin, and belongs to the technical field of medicinal chemistry. According to the invention, 27 kinds of cytochalains are separated from a soil fungus Xylaria sp. Y01, wherein the 27 kinds of cytochalains comprise 13 kinds of newly discovered compounds. It is explored whether these compounds have an inhibitory effect on the release of pro-inflammatory cytokines. The result shows that the compound 22 shows a remarkable anti-inflammatory effect on RAW264.7 cells by up-regulating the expression of SOCS3. Therefore, the compound 22 is expected to become a potential candidate drug for developing drugs for treating inflammatory diseases.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, and more particularly to a method based on soil fungi. Xylaria Cytochalasin of sp. Y01 and its application. Background Technology

[0002] Cytochalasin is a diverse class of fungal metabolites with a wide range of biological functions, derived from the combined action of polyketide compounds and nonribosomal peptide synthases (PKS-NRPS). Since 1966, more than 500 cytochalasin species have been discovered, primarily isolated from fungi in the genera *Anthracis*, *Mucor*, *Aspergillus*, *Penicillium*, and *Zygosporum*. In addition to expanding their chemical diversity, significant progress has been made in recent decades in elucidating their biological activities, molecular targets, and mechanisms.

[0003] Soil fungi are a rich source of secondary metabolites, exhibiting unique chemical diversity. Studies have shown that these fungi can produce a variety of novel metabolites, such as pyridone derivatives, cytochalasin, macrocyclic trienones, and condensed phenolic acid derivatives. Since the first report of cytochalasin (cytochalasin A and B), a variety of cytochalasin analogs have been identified, possessing diverse activities including cytotoxicity, antibacterial properties, antifungal effects, and immunosuppressive capabilities, and have been extensively studied. Summary of the Invention

[0004] The purpose of this invention is to provide a soil fungus-based Xylaria The study explores the cytochalasin of sp. Y01 and its applications to enrich the existing types of cytochalasin and address the issue of chemical diversity.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a soil fungus-based Xylaria sp. Y01's cytochalasin comprises compounds 1 to 13 with the following structural formulas: ; Where Δ a,b This indicates that the double bond is located between the carbon atom at position a and the carbon atom at position b.

[0006] The present invention also provides the above-mentioned soil fungi-based Xylaria Application of sp. Y01 cytochalasin in the preparation of anti-inflammatory drugs.

[0007] The present invention also provides the use of compounds 14 to 27 in the preparation of anti-inflammatory drugs, wherein the structural formulas of compounds 14 to 27 are as follows: .

[0008] The beneficial effects of this invention are: This invention is derived from soil fungi Xylaria Twenty-seven cytochalasin compounds (1-27) were isolated from sp. Y01, including 13 newly discovered compounds (1-13). The molecular structures of these new compounds were resolved using a combination of spectroscopic analysis and quantum chemical calculations. The inhibitory effects of most of these 27 isolates on nitric oxide (NO) production in mouse RAW264.7 macrophages stimulated by lipopolysaccharide (LPS) were evaluated. The results showed that compound 22 exhibited a significant anti-inflammatory effect on RAW264.7 cells by upregulating SOCS3 expression. Therefore, compound 22 shows promise as a potential drug candidate for the development of treatments for inflammatory diseases. Attached Figure Description

[0009] Figure 1 For compounds 1-13 1 H- 1 Schematic diagram of the correlation between H COSY and key HMBC; Figure 2 The key nuclear Oferheim effect (NOE) correlations of compounds 1-12 are shown in the lowest energy conformation diagram; Figure 3 Experimental and calculated ECD curves for compounds 1-12 and their enantiomers; Figure 4 The HPLC chromatograms are for compounds 12 and 13. Figure 5 Experimental and calculated ECD curves of compound 13 and its enantiomers are key values ​​for NOE correlation. Figures 6 to 17 Compounds 1 to 11 are respectively 1 H NMR spectrum; Figure 18 To detect the levels of TNF-α, MCP-1, and IL-6 in cells treated with compound 12 (A), compound 21 (B), and compound 22 (C) using an ELISA kit, cells treated with blank carrier solution were used as blank controls. Figure 19Compound 22 exhibits anti-inflammatory effects on RAW264.7 cells by increasing SOCS3 expression. (A) Volcano plot of differentially expressed genes (DEGs) in RAW264.7 cells between the 22-treated and control groups. The X-axis represents the log2 fold change, and the Y-axis represents the -Log10 false discovery rate (FDR). Blue indicates downregulated expression, and red indicates upregulated expression. (B) KEGG pathway enrichment analysis of DEGs. (C) Heatmap of DEGs in RAW264.7 cells between the 22-treated and control groups. (DF) RT-qPCR results show changes in SOCS3, Cxcl10, and TNF-α mRNA levels in RAW264.7 cells after 22 treatment. (G) Western blot analysis shows upregulation of SOCS3 protein levels in RAW264.7 cells after 22 treatment. Detailed Implementation

[0010] This invention provides a soil fungus-based Xylaria sp. Y01's cytochalasin comprises compounds 1 to 13 with the following structural formulas: ; Where Δ a,b This indicates that the double bond is located between the carbon atom at position a and the carbon atom at position b.

[0011] The present invention also provides the above-mentioned soil fungi-based Xylaria Application of sp. Y01 cytochalasin in the preparation of anti-inflammatory drugs.

[0012] The present invention also provides the use of compounds 14 to 27 in the preparation of anti-inflammatory drugs, wherein the structural formulas of compounds 14 to 27 are as follows: .

[0013] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0014] Example 1

[0015] Compound 1 was obtained as a white powder. UV spectroscopy showed absorption peaks at 210 and 260 nm. IR spectroscopy revealed ester groups (1733 cm⁻¹). -1 ) and hydroxyl (3346 cm) -1 The characteristic absorption band of compound 1 was determined by HRESIMS measurements to be C10. 31 H 41 NO8 ( m / z 578.2724 [M+Na] +Δ -0.2 ppm), with an IHD (hydrogen deficiency index) of 12.

[0016] 1 H and 13 1C NMR data showed that 1 had signals typical of the cell wall cytoskeleton, including δ H Monosubstituted phenyl groups at positions 7.13 (2H, d, J = 7.7 Hz), 7.30 (2H, t, J = 7.7 Hz), and 7.23 (1H, t, J = 7.7 Hz); δ H A methyl doublet at position 0.79 (3H, d, J = 6.5 Hz); δ H Two groups of methylene groups at positions 2.86 (1H, dd, J = 13.4, 6.8 Hz) and 2.94 (1H, dd, J = 13.4, 8.4 Hz); δ H Two methylene groups at positions 1.85 (1H, overlapping) and 1.45 (1H, ddd, J=12.5, 12.1, 12.1 Hz); δ C Ketone carbon at position 212.4; δ C An amide group at position 175.2; δ C An ester carbonyl group at position 172.1; δ C Two quarter-carbon double bonds at positions 125.0 and 133.0; δ C Five methyl singlets at positions 14.2, 17.4, 21.2, 25.3, and 59.9; δ C Two tertiary carbons at positions 50.4 and 83.6 (sp3 hybridization); δ C The methyl group at position 59.4, which links to a nitrogen atom; δ C The four oxacyclic propane carbons at positions 70.7, 71.5, 72.2, and 84.5; and δ C Five high-field methyl carbon signals at positions 36.1, 39.5, 41.6, 43.2, and 48.6. From... 1 H- 1 In the H COSY spectrum, three spin-spin coupled systems can be observed: H2-10 / H-3( / 2-NH) / H-4, H-25 / H-26 / H-27 / H-28 / H-29, and H-7 / H-8 / H-13 / H-14(H2-15 / H-16 / H3-23) / H-20 / H-21(H-19). Figure 1 HMBC correlations between NH and C-3 / C-4 / C-9, H-3 and C-1, H-4 and C-1 / C-9, H-3 and C-9, H-3 and C-4 / C-6, and H-3 and C-5 / C-6 / C-7. Figure 1The structural connection between rings A and B was confirmed. The HMBC correlation between 7-OH and C-6 / C-7 / C-8, H-21 and C-9, H-3 and C-16 / C-17, and H-3 and C-17 / C-18 / C-19, combined with... 1 H- 1 H COSY correlation revealed structural connections between rings B, C, and D. H2-10 and C-24 (δ...) C 137.7) and C-25 / C-29 (δ C The HMBC correlation of 129.2) indicates that the phenyl substitution is at position 10. Therefore, the planar structure of compound 1 is as follows: Figure 1 As shown.

[0017] Given that rings B, C, and D are typical cyclohexane structures, the J coupling constants between ortho-protons provide important evidence for determining their stereochemical orientation. The coupling constants of H-7 (9.7 Hz), H-8 (10.0 Hz, 9.7 Hz), H-13 (10.4 Hz, 10.0 Hz), H-19 (10.5 Hz), H-20 (12.3 Hz, 10.5 Hz, 2.1 Hz), and H-21 (2.1 Hz) indicate that they are in the upright, upright, upright, upright, upright, and prostrate positions, respectively. The NOE correlations between H-3 / H3-11, H-4 / H-21, and H-21 / H2-10 show that H-3 is located on the same side of ring A as ring B, while H-4, H2-10, and H-21 are located on the other side. H-8 / H-14, H-14 / H-16, H-16 / H-19, H-19 / H3-22, and H-7 / H-13, H-13 / H a -15, H a The NOE cross peaks between -15 and H-20 indicate that they are in the same orientation. Since H-7 and H-8 are upright and point in opposite directions, the orientations of the two groups of hydrogen atoms are also opposite. Therefore, their relative configurations are as follows: Figure 2 As shown.

[0018] The absolute configuration of compound 1 was determined by ECD calculations. Figure 3 At the ωB97X-D / TZVP (IEFPCM, acetonitrile) level, the ECD curves for 3S, 4R, 7S, 8S, 9R, 13S, 14R, 16S, 17S, 19R, 20S, and 21R-1 showed excellent agreement with experimental ECD data. Therefore, the structure of compound 1 was fully determined and named curtachalasin Y1.

[0019] Example 2

[0020] Compound 2 was obtained as a white powder, and its molecular formula was determined to be C.31 H 41 NO8, via mass spectrometry ( m / z 1133.5575[2M+Na] + The concentration (Δ 0.0 ppm) was determined, the same as that of compound 1. The concentration of compound 2... 1 H and 13 The C10 NMR data showed a similar signal to compound 1, indicating the presence of a monosubstituted phenyl group, ring A, and ring D (Tables 1 and 2). Furthermore, HSQC, 1 H- 1 H COSY and HMBC spectra (2DNMR data) indicate that they have the same planar structure. Figure 1 The coupling constant of H-13 (10.3, 10.3 Hz) indicates that it is in an upright position. H-7 / 13-OCH3, 13-OCH3 / H eq -15 and H eq The NOE cross peaks between -15 / H3-23 confirm their similar orientation. 13 C10 NMR data showed that the chemical shifts of compound 2 at C-1, C-5, C-7, C-12, and C-13 differed by more than 2 ppm. Therefore, we verified their relative configurations using GFN2 NMR. Comparison of experimental data and their respective GFN2 NMR calculations for compounds 1 and 2 confirmed the above inference. At the ωB97X-D / TZVP (IEFPCM, methanol) level, the calculated ECD curves for 3S, 4R, 7R, 8R, 9R, 13S, 14R, 16S, 17S, 19R, 20S, and 21R-2 were in excellent agreement with the experimental ECD. Therefore, its structure was fully determined and named curtachalasin Y2.

[0021] Example 3

[0022] Compound 3 was obtained as a white powder with the molecular formula C. 31 H 41 NO8, as determined by HRESIMS ( m / z 556.2914 [M+H] + Δ +1.6 ppm), the same as compounds 1 and 2. In 1 H and 13 In their 10⁻¹⁵ NMR spectra, they also show similar signals, except for 3 at C⁻⁷ and C⁻¹³. 13 δ¹⁻¹ NMR chemical shifts were observed. C 76.5 and 66.7 (1 for δ) C 70.7 and 84.5, 2 for δ C67.8 and 78.8). These significant differences indicate that they have different structures at these positions. HMBC correlation between OCH3 and C-7 ( Figure 1 This indicates that the methoxy group is substituted at C-13. 1 H- 1 H COSY correlation and coupling constant indicate that, located at δ H 5.93 ( 1 The hydroxyl group (H, d, J = 11.5 Hz) is linked to C-13. The signals for the remaining group linkages in the 2D NMR are consistent with those of compound 1. Linkages are achieved via two sets of protons: H-8 / H-14, H-14 / H-16, H-16 / H-19, and H-19 / H-3-22, and 7-OCH3 / H-13, H-13 / H... eq -15 and H eq NOE correlation between -15 / H3-23, relative configuration as follows Figure 1 As shown. In M062X / 6-311+G The ECD curves of 3S, 4R, 7S, 8S, 9R, 13R, 14R, 16S, 17S, 19R, 20S, 21R-3 calculated at the (IEFPCM, acetonitrile) level matched well with the experimental ECD. Therefore, the structure of compound 3 was fully determined and named curtachalasin Y3.

[0023] Example 4

[0024] Compound 4 was obtained as a white powder with the molecular formula C. 31 H 41 NO8, determined by HRESIMS ( m / z 1133.5592 [2M+Na] + (Δ +1.5ppm), the same as compounds 1-3. 1 H / 13 C NMR, HSQC, 1 H- 1 H COSY and HMBC spectra confirmed that compounds 3 and 4 have the same planar structure. Figure 1 However, they exhibit significant chemical shift differences (>5 ppm) at C-7 and C-13 (Table 2). The coupling constants of H-7 (9.8 Hz) and H-13 (9.5, 9.5 Hz) indicate that the coupled protons H-7 and H-13 are in an upright position. In contrast, the H-13 proton in compound 3 occupies a flat position. The orientation of 13-OH changes ( Figure 2 This may therefore affect the chemical shifts at C-7 and C-13. This hypothesis is consistent with the GFN2 NMR findings of compounds 3 and 4. 13The CNMR calculation results are consistent. Furthermore, H-7 / H-13 and H-13 / H... ax The NOE cross peaks between -15 and 7-OCH3 / 13-OH confirm the relative configuration of compound 4. Figure 2 (In M062X / 6-311+G) The ECD curves of 3S, 4R, 7S, 8S, 9R, 13S, 14R, 16S, 17S, 19R, 20S, and 21R-4 calculated at the (IEFPCM, acetonitrile) level matched well with the experimental ECD. Therefore, the structure of compound 4 can be fully determined and named curtachalasin Y4.

[0025] Example 5

[0026] Compound 5 was obtained as a white powder with the molecular formula C. 30 H 39 NO8, determined by HRESIMS ( m / z 542.2752 [M+H] + Δ +0.6 ppm). 1 H and 13 C10 NMR data indicate that compound 5 has one less methoxy group signal than compound 3, and the chemical shift at C-7 is shifted 10.3 ppm towards the higher field. The remaining planar structure of compound 5 is the same as that of compound 2, as shown below. Figure 1 As shown. The coupling constants of H-7 (J = 10.7 Hz) and H-8 (J = 10.7, 3.3 Hz) support the upright configuration of these two protons. Furthermore, the NOE correlation of H-7 / 13-OH indicates that they are located on the same side of the ring system (…). Figure 2 Compound 5 is considered to be its isomer at position 13, compared to the known compound 14. We compared them in the same solvent (CDCl3). 13 The 10⁻¹⁴ NMR chemical shifts showed significant differences near the isomerization sites. We further calculated their chemical shifts using GFN₂NMR. 13 C10 NMR chemical shifts confirmed the above structural assignments. ECD calculations at the theoretical level of B3LYP / TZVP (IEFPCM, methanol) further determined the absolute configurations of 5 to be 3S, 4R, 7S, 8S, 9R, 13R, 14R, 16S, 17S, 19R, 20S, 21R (…). Figure 3 ).

[0027] Example 6

[0028] Compound 6 was obtained as a white powder with the molecular formula C6. 32 H 43NO8, determined by HRESIMS ( m / z 592.2907 [M+Na] + Δ +4.4ppm). Compared to compound 4, 6 1 1H NMR data showed an additional ethyl group, characterized by δ H The methyl group at 1.09 (3H, dd, J = 7.0, 7.0 Hz) and δ H The methylene group at position 3.53 ( 1 H, dq, J = 9.2, 7.0 Hz) and at 3.64 1 H, dq, J = 9.2, 7.0 Hz. Furthermore, the chemical shift of C-7 differs significantly from that of compound 4, shifting downwards by 8.9 ppm. The HMBC correlation between the ethyl group and C-7 indicates that the ethoxy group is attached to C-7 ( Figure 1 The NOE correlation between H-4 and H-8 confirms their coplanar orientation on ring B, with both protons in an upright position. Figure 2 The coupling constant (2.1 Hz) between H-7 and H-8 indicates that H-7 is in a flat position. The remaining planar structure and relative configuration of 6 are the same as those of 4. Figure 1 Finally, ECD calculations at the theoretical level of ωB97X-D / TZVP (IEFPCM, methanol) determined the absolute configurations of 6 to be 3S, 4R, 7R, 8S, 9R, 13S, 14R, 16S, 17S, 19R, 20S, 21R (…). Figure 3 ).

[0029] Example 7

[0030] Compound 7 was obtained as a white powder with the molecular formula C. 30 H 43 NO8, determined by HRESIMS ( m / z 1069.5055 [2M+Na] + Δ +2.1ppm). Compared to compound 2, 7 1 H and 13 C10 NMR data indicate that compound 7 lacks one methoxy group and two methylene groups, but instead has a δ-ray concentration of 100 nm. C An additional double bond signal was observed at 120.6 and 134.5. 2D NMR analysis confirmed that 7 is a 13,14-dehydrogenated product derived from 2. ECD calculations at the theoretical level of B3LYP / TZVP (IEFPCM, acetonitrile) further determined the absolute configuration of 7. Figure 3 ).

[0031] Example 8

[0032] Compound 8 was obtained as a white powder with the molecular formula C. 30 H 37 NO8, determined by HRESIMS ( m / z 1101.4925 [2M+Na] + Δ -0.5ppm). Compared with compound 4, 8 1 H and 13 C NMR data indicate that it lacks a methoxy group, and in δ C An additional carbonyl group was observed at 199.3. The HMBC correlation between these H3-12 / H-8 / H-13 and C-7 indicates the position of the carbonyl group. 2D NMR data confirm that compound 8 is the 7-oxidation product of 4 (…). Figure 1 The absolute configuration of 8 was confirmed by ECD calculations at the theoretical level of B3LYP / TZVP (IEFPCM, methanol). Figure 3 ).

[0033] Example 9

[0034] Compound 9 was obtained as a white powder with the molecular formula C. 30 H 37 NO7, determined by HRESIMS ( m / z 1069.5045 [2M+Na] + (Δ +1.1ppm). Compared to 8, it has one less oxygen atom. (Through...) 1 H and 13 Comparison of C NMR data, 9 in δ C There is an additional methylene group at 26.6 (C-13). 2D NMR analysis ( Figure 2 The results showed that the remaining structure of compound 9 was identical to that of compound 8, confirming it as the product of deoxygenation at position 13 of compound 8. ECD calculations at M062X / 6-311+G... (IEFPCM, acetonitrile) At the theoretical level, the absolute configurations of 9 are determined to be 3S, 4R, 9R, 15R, 16S, 17S, 19R, 20S, 21R. Figure 3 ).

[0035] Example 10

[0036] Compound 10 was obtained as a white powder with the molecular formula C0. 31 H 35 NO8, determined by HRESIMS ( m / z 572.2257 [M+Na] +The concentration of Δ +0.3 ppm indicates that its IHD is 15. The UV spectrum of compound 10 in methanol shows maximum absorption peaks at 230 and 365 nm, suggesting the presence of multiple conjugated structures within its molecular framework. 1 H, 13 C10 NMR, DEPT, and HSQC spectra revealed characteristic signals, including the signal of a monosubstituted benzene ring located at δ1000 ppm. H 7.22 (2H, overlap), 7.30 (2H, t, J = 7.6Hz), 7.23 (1H, overlap); aldehyde group located at δ H 10.15 (1H, s); the methoxy group is located at δ H 3.40 (3H, s); a methyl bimodal peak is located at δ H 1.06 (3H, d, J = 7.0 Hz); the three methyl groups attached to the double bond are located at δ H 2.10 (3H, s), 2.30 (3H, d, J = 1.7 Hz), 2.31 (3H, s); amide group located at δ C 173.2; One ester group is located at δ C 171.6; Three double bonds are located in δ C 128.1, 128.7, 131.2, 137.4, 137.4, 147.5; the methylene group is located at the δ C 44.2. 1 H- 1 H COSY and HMBC signals indicate a basic framework for the 5 / 6 / 6 / 6 fused ring system, as shown in compounds 1-9. Figure 1 These HMBC correction signals H-7 to C-5 / C-6 / C-9, H-13 to C-8 / C-9 / C-14 / C-15 / C-20, H-21 to C-9, and H3-12 to C-5 / C-6 / C-7 validate the planar structure of rings B and C. The chemical shift of the aldehyde group shifts to a higher field, indicating its conjugation with the double bond. The HMBC correlation of the aldehyde group with C-4 / C-5 / C-6 determines the position of the aldehyde group. The chemical shift at C-15 and the HMBC correlation of the methoxy group with C-15 indicate the position of the methoxy group (…). Figure 1 The rest of the planar structure is consistent with compounds 1-9. Figure 1 Furthermore, the NOE correlation between H-16 and H-19 indicates that H-16 and H-19 are located on the same side of ring D and are both in an upright position. Figure 2 The coupling constant (J = 2.8 Hz) between H-15 and H-16 indicates that H-15 is in a flat position. The NOE correlation between H-4 and H2-10 / H-21 indicates that both benzyl and H-4 are located on the same side of ring A. Figure 2The relative configurations of compound 10 shown are obtained by M062X / 6-311+G ECD calculations at the theoretical level for (IEFPCM, methanol) determined its absolute configuration to be 3S, 4R, 9R, 15R, 16S, 17S, 19R, 20S, 21R. Figure 3 ).

[0037] Example 11

[0038] Compound 11 was obtained as a white powder with the molecular formula C11. 30 H 37 NO7, via HRESIMS ( m / z 546.2460 [M+Na] + Determined by Δ -0.6ppm. 1 H and 13 The C NMR spectrum signal indicates that it has the same basic skeleton as compounds 1-10. Figure 1 Compound 11 contains an sp... 2 Methylene, signal located at δ H 5.10 ( 1 H, brs), 5.15 ( 1 H, brs) and δ C 113.4. HMBC correlations between H2-12 and C-5 / C-6 / C-7, and between H-7 and C-5 / C-6 / C-9, and between H2-10 / H-3 / H-4 / H-5 / H3-11. 1 H- 1 H COSY correlation revealed the structure of rings A and B ( Figure 1 The relative configurations of ring C and D supported by 2D NMR data are consistent with those of compound 3. Figure 1 The NOE correlation between H-3 and H3-11 indicates that H-3 and H3-11 are located on the same side of ring B. Given the NOE correlation between H-14 and H-19 and the coupling constant of H-19, they are both located on the same side of ring D. Figure 2 Furthermore, the coupling constant between H-13 and H-14 indicates that H-13 is in a flat position. The relative configuration of 11 has been fully determined. Figure 2 (via M062X / 6-311+G) Based on ECD calculations at the theoretical level (IEFPCM, acetonitrile), the absolute configuration of 11 was determined to be 3S, 4R, 5S, 9R, 13R, 14R, 16S, 17S, 19R, 20S, 21R (…). Figure 3 ).

[0039] Example 12

[0040] Compound 12 was obtained as a white powder with the molecular formula C12. 30 H 39 NO8, determined by HRESIMS ( m / z 542.2748 [M+H] + (Δ -0.2ppm), with one more oxygen atom than compound 11. Compounds 12 and 11 have very similar structures, except that they lack a double bond and each contains two additional sp atoms. 3 methylene group (δ C 43.1, 69.4), this is through their 1 H and 13 The comparison of C12 NMR data confirmed this. Two-dimensional NMR confirmed that 12 is substituted with a hydroxyl group at the C-7 position. Figure 1 The coupling constant (10.5 Hz) of H-7 indicates that both H-7 and H-8 are in an upright position. The remaining relative configurations are consistent with compound 11, a fact supported by the coupling constant and NOE correlation. Figure 2 The absolute configuration of 12 was confirmed by ECD calculations at the theoretical level of B3LYP / TZVP (IEFPCM, acetonitrile). Figure 3 ).

[0041] Example 13

[0042] Compound 13 was obtained as a white powder with the molecular formula C13. 30 H 39 NO8, via HRESIMS ( m / z 542.2739[M+H] + The concentration was determined by Δ -1.8 ppm. Compounds 13 and 12 have the same planar structure, which was determined by... 1 H / 13 C NMR, HSQC, 1 H- 1 The HPLC chromatograms confirmed this. However, they are two different compounds with different retention times in the HPLC chromatograms. Figure 4 Their main difference lies in the methyl carbon at the C-13 position (δ¹³ of C). C δ 73.8,12 C The coefficient of performance (J = 68.8) indicates that epimerization occurred at this position. The coupling constant of H-13 (J = 10.0, 10.0 Hz) indicates that H-8, H-13, and H-14 are all in the upright position (Table 4), which is confirmed by the NOE correlation observed between H-7 and H-13. The remaining relative configurations are consistent with those of compound 12, which is also supported by the NOE correlation. Figure 5The absolute configuration of compound 13 was confirmed by ECD calculations at the theoretical level of B3LYP / TZVP (IEFPCM, methanol). Figure 5 ).

[0043] Table 1. Compounds 1-4 in CDCl3 1 1H NMR data (600 MHz) (δ in ppm, J in Hz)

[0044] Table 2. Compounds 1-4 in CDCl3 13 C NMR data (150 MHz) (δ in ppm)

[0045] Table 3. Compounds 5-7 1 H NMR data (600 MHz) and 13 C NMR data (150 MHz) (δ in ppm, J in Hz)

[0046] a Measured in units of pyridine-d5; b Measured in units of CD3Cl; c Measured in CD3OD units; d Determined by chemical shift value.

[0047] Table 4. Compounds 8 and 9 in CDCl3 1 H NMR data (600 MHz) and 13 C NMR data (150 MHz) (δ in ppm, J in Hz)

[0048] Table 5. Compounds 10-13 1 1H NMR data (600 MHz) (δ in ppm, J in Hz)

[0049] a The determination was performed using CDCl3 as the solvent; b The determination was performed using CD3OD as the solvent; c Determined based on chemical shift value.

[0050] Table 6. Compounds 10-13 13C NMR data (150 MHz) (δ in ppm)

[0051] a The determination was performed using CDCl3 as the solvent; b The determination was performed using CD3OD as the solvent; c Determined based on chemical shift value.

[0052] Anti-inflammatory activity tests of the above compounds: To determine the anti-inflammatory activity of these cytochalasins, the inhibitory effects of compounds (1, 5, 7, 8, 12, 13-16, and 18-27) in adequate amounts on NO production in an LPS-induced RAW264.7 cell model were evaluated, with dexamethasone (DEX) as a positive control. First, their cytotoxicity to RAW264.7 cells was determined using a CCK-8 assay. Sixteen cytochalasins (1, 5, 7, 8, 12, 13-16, 19-23, 26, and 27) showed cell viability exceeding 90% after treatment with 30 μM and were used for further evaluation. Their inhibitory activity on NO production was assessed by evaluating nitrate / nitrite levels in the LPS-induced RAW264.7 cell supernatant. Compared to the solvent-treated LPS group, treatment with compounds 12, 21, and 22 showed inhibitory effects on NO levels in RAW264.7 cells, with half-maximal inhibitory concentrations (WMCs) of 43.8, 55.4, and 17.8 μM, respectively. Notably, compound 22 was close to the effect of DEX (WMC = 14.9 μM). The WMCs of the other compounds all exceeded 60 μM (Table 7).

[0053] Previous studies have shown that RAW264.7 cells release large amounts of pro-inflammatory cytokines, including IL-6, MCP-1, and TNF-α, during LPS-mediated inflammatory responses. Therefore, this study investigated whether these compounds inhibited the release of pro-inflammatory cytokines. Our ELISA results showed that treatment with compounds 12, 21, and 22 significantly reduced the levels of IL-6, MCP-1, and TNF-α. Figure 18 This confirms their anti-inflammatory effects.

[0054] Table 7. Half-inhibitory concentrations (WICs) of the tested compounds on the inhibitory effect of LPS-induced NO production in RAW264.7 macrophages.

[0055] This study collected soil samples from Yunnan, China, from which *Anthracis* strain Y01 was isolated. Sample processing and fungal identification methods followed our previously published literature 19 (Journal of Molecular Structure, 2025, 1321, 140274). The mycelium of strain Y01 was placed in SDA liquid medium and cultured at 25 °C for 10 days, then inoculated into 500 mL Erlenmeyer flasks of rice culture medium and cultured for another 40 days. After harvesting 10 kg of rice culture containing strain Y01, extraction was performed with 20 L of ethyl acetate at room temperature. The crude extract was obtained by vacuum evaporation, partitioned five times with ethyl acetate and water, and concentrated under reduced pressure to obtain a brown extract (150 g). The crude extract was adsorbed onto macroporous resin D101 and packed into a 20 cm × 30 cm column of the same resin. The column was washed sequentially with 0% (discarded), 90%, and 100% methanol aqueous solutions to obtain three fractions: fractions A and C. After silica gel column chromatography, fraction A was eluted with a gradient of petroleum ether (PE)-ethyl acetate (EA)-methanol (volume ratio 1:0:0 to 0:0:1) to finally obtain five corresponding fractions (A1-A5).

[0056] Fr. A1 was purified by a semi-preparative HPLC system (flow rate 2.5 or 3 mL / min) to obtain 12 (26.6 mg, 40% acetonitrile). t R = 22.0 min, SinoChrom ODS-AP: 10.0 mm × 250 mm, 5 μm), 13 (68.2 mg, 50% methanol, t R = 35.0 min, Supersil C8: 10.0 mm × 250 mm, 5 μm) and 16 (4.1 mg, 67% acetonitrile, tR = 20.0 min, Supersil C8: 10.0 mm × 250 mm, 5 μm).

[0057] Fr. A2 was fractionated by microporous resin (MCI) and eluted with methanol-H2O (v / v 20:80, 40:60, 60:40, 80:20, 100:0, 20 mL / min) to obtain five subfractions (A2a-A2e).

[0058] The remaining Fr. A2a was purified by a semi-preparative HPLC system (flow rate 2.5 or 3 mL / min) to obtain 5 (50.3 mg, 59% methanol). t R= 30 min, SinoChrom ODS-AP: 10.0 mm × 250 mm, 5 μm), 6 (3.6 mg, 63% methanol, t R = 31.5 min, SinoChrom ODS-AP: 10.0 mm × 250 mm, 5 μm), 7 (9.4 mg, 31% acetonitrile, t R = 47 min, Supersil C8: 10.0 mm × 250 mm, 5 μm), 8 (5.3 mg, 59% methanol, t R = 41.0 min, SinoChrom ODS-AP: 10.0 mm × 250 mm, 5 μm), 14 (8.3 mg, 33% acetonitrile, t R = 38 min, Supersil C8: 10.0 mm × 250 mm, 5 μm), 15 (5.0 mg, 33% acetonitrile, t R = 36.5 min, Supersil C8: 10.0 mm × 250 mm, 5 μm) and 17 (1.2 mg, 59% methanol, t R = 33 min, SinoChrom ODS-AP: 10.0 mm × 250 mm, 5 μm).

[0059] The remaining Fr.A2b was sequentially purified using a semi-preparative HPLC system (flow rate 2.5 or 3 mL / min) to obtain 2 (1.1 mg, 39% acetonitrile, t R = 22.0 min, Supersil C8: 10.0 mm × 250 mm, 5 μm), 3 (1.2 mg, 68% methanol, t R = 33.0 min, SinoChrom ODS-AP: 10.0 mm × 250 mm, 5 μm), 4 (0.9 mg, 72% methanol, t R = 25.0 min, SinoChrom ODS-AP: 10.0 mm × 250 mm, 5 μm), 9 (1.0 mg, 39% acetonitrile, t R= 22.0 min, SinoChrom ODS-AP: 10.0 mm × 250 mm, 5 μm), 10 (0.8 mg, 54% methanol, t R =28.0 min, SinoChrom ODS-AP: 10.0 mm × 250 mm, 5 μm) and 11 (1.3 mg, 68% methanol, t R =29.0 min, SinoChrom ODS-AP: 10.0 mm × 250 mm, 5 μm).

[0060] The remaining Fr.A2c was purified by a semi-preparative HPLC system (flow rate 2.5 mL / min) to give compound 1 (10.7 mg, 34% methanol). t R = 23.0 min, SinoChrom ODS-AP: 10.0 mm × 250 mm, 5 μm).

[0061] The inventors performed RNA sequencing-based transcriptome analysis on RAW264.7 cells treated with compound 22 to preliminarily explore the underlying mechanisms. This high-throughput method enabled us to identify genes and pathways altered after compound 22 treatment. The volcano plot shows (…). Figure 19 A) A total of 217 differentially expressed genes (DEGs) were identified between compound 22-treated and untreated RAW264.7 cells, of which 126 genes were upregulated and 91 genes were downregulated. KEGG pathway enrichment analysis showed that the DEGs identified in compound 22-treated RAW264.7 cells (compared to untreated cells) were associated with the TNF signaling pathway, which is closely involved in the inflammatory response. For example, DEGs such as CXC motif chemokine ligand 10 (CXCL10) and CC motif ligand 2 (CCL2) were significantly downregulated in compound 22 treatment, while cytokine signaling inhibitor 3 (SOCS3) was significantly upregulated. Figure 19 B and 19C). Further mRNA expression analysis showed that, compared with the control group, compound 22 treatment increased SOCS3 mRNA expression in RAW264.7 cells (B and 19C). Figure 19 D, P < 0.01). Furthermore, with increasing treatment dose of compound 22, the production of Cxcl10 and TNF-α in RAW264.7 cells decreased (D, P < 0.01). Figure 19E and 19F, P < 0.001). To further validate the results, we performed Western blot analysis, which showed that compound 22 treatment increased SOCS3 protein expression in RAW264.7 cells in a dose-dependent manner compared with the LPS-induced control group. Figure 19 G). Previous studies have shown that CXCL10 plays a key role as an inflammatory mediator, while SOCS3 acts as an anti-inflammatory factor, negatively regulating TNF-α activation. Therefore, compound 22 may inhibit the TNF signaling pathway and reduce the inflammatory response in RAW264.7 cells through a mechanism involving CXCL10 and SOCS3.

[0062] This invention is derived from soil fungi Xylaria Twenty-seven cytochalasin compounds (1-27) were isolated from sp. Y01, including 13 newly discovered compounds (1-13). The molecular structures of these new compounds were resolved using a combination of spectroscopic analysis and quantum chemical calculations. The inhibitory effects of most of these 27 isolates on nitric oxide (NO) production in mouse RAW264.7 macrophages stimulated by lipopolysaccharide (LPS) were evaluated. The results showed that compound 22 exhibited a significant anti-inflammatory effect on RAW264.7 cells by upregulating SOCS3 expression. Therefore, compound 22 shows promise as a potential drug candidate for the development of treatments for inflammatory diseases.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A soil fungus-based Xylaria sp. Y01's cytochalasin, characterized in that, Compounds 1 to 13 include the following structural formulas: ； wherein Δ a,b represents a double bond between the carbon atom in position a and the carbon atom in position b.

2. The soil fungus-based method according to claim 1 Xylaria Application of sp. Y01 cytochalasin in the preparation of anti-inflammatory drugs.

3. A method based on soil fungi Xylaria The application of sp. Y01's cytochalasin in the preparation of anti-inflammatory drugs is characterized by, The cytochalasin comprises compounds 14 to 27 with the following structural formulas: 。

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